This is a request for renewal of a research program on thiamin diphosphate-dependent enzymes, principally on the E. coli and human pyruvate dehydrogenase multienzyme complexes. These complexes are at a key junction in metabolism of virtually all cells, converting the product of glycolysis pyruvic acid to acetyl coenzyme A at the entry to the Krebs cycle (also known as the tricarboxylic acid or citric acid cycle). Goals for the next phase of the project are: (1) Determination of rate-limiting steps, states of ionization and tautomerization of enzyme-bound thiamin-related intermediates, with the intent to compare these properties in isolated components of the complexes to those in the 4,600,000 and 10,000,000 Da complexes. In innovation requiring de novo total synthesis of specifically labeled thiamins, the PI proposes to use solid state NMR, as well as advanced solution NMR methods to accomplish the goals. (2) Exploration of the hypothesis that the mobility of active center loops in thiamin enzymes is correlated with catalysis. With methods published by the PI in 2008, key loops on the E. coli complex's E1 and E2 components have been identified for these studies, including the lipoyl domain of the E2 component, which 'visits' the active centers of all three components. (3) Examination of the structural and functional consequences of assembly in the bacterial and human pyruvate dehydrogenase complexes. In the E. coli complex, the issue to be resolved is whether the same region(s) of the E2 component recognizes the E1 and E3 components, to address the hypothesis that the loci of recognition of E2 for E1 or E3 are different. In the human complex, we wish to determine the regions of E2 component interacting with the E1 component, never accomplished before The methods used by the PI could answer questions that no other current methodology can, such as the definition of proton positions on the 4'-aminopyrimidine ring of the coenzyme at distinct intermediates along the reaction coordinate, a key issue in understanding proton transfers. A group of outstanding collaborators have been recruited for experimental expertise not available in the PI's laboratory, among them M. Patel, a CoPI selected for expertise on the human enzyme, and W. Furey, a long-term collaborator in a string of structure determinations. Results from two complexes will enable the PI to draw general conclusions regarding the entire superfamily of such enzymes, as already demonstrated in several of his recent publications. PUBLIC HEALTH RELEVANCE: This is a request for renewal of a research program on thiamin diphosphate dependent enzymes, principally on the E. coli and human pyruvate dehydrogenase multienzyme complexes. These complexes are at a key junction in metabolism of virtually all cells, converting the product of glycolysis pyruvic acid to acetyl coenzyme A at the entry of the Krebs cycle (also known as the tricarboxylic acid or citric acid cycle). Goals for the next phase of the project include: (1) Determination of rate-limiting steps, states of ionization and tautomerization of enzyme-bound thiamin-related intermediates, with the intent to compare these properties in isolated components of the complexes to those in the 4,600,000 and 10,000,000 Da complexes. (2) Explore the hypothesis that the mobility of active center loops in thiamin enzymes is correlated with catalysis. (3) Examine the structural and functional consequences of assembly in the bacterial and human pyruvate dehydrogenase complexes. In the E. coli complex, the issue to be resolved is whether the same region(s) of the E2 component recognize the E1 and E3 components, to address the hypothesis that the loci of recognition of E2 for E1 or E3 are different. In the human complex, the issue to be addressed is determination of the regions of E2 component interacting with the E1 component, never accomplished before. Recent evidence indicates involvement of the kinase-dependent control of the human pyruvate dehydrogenase complex activity in both diabetes and cancer making structural studies of the human E2 component particularly timely. The studies proposed are intertwined with structural studies carried out in collaboration with highly productive and prominent groups.